In the mid-1970's, it was discovered that opium alkaloids such as morphine and heroin bind to specific receptors in brain and other tissues. The specific binding reaction was found to be prerequisite to the production of the characteristic biological effects of analgesia and euphoria for which the opiates are well known. The findings led to the discovery of endogenous substances which also bind to the opiate receptors. These naturally occurring compounds are termed endorphins, for endogenous morphine-like substances. A number of such compounds have been isolated and characterized and shown to be peptides having similarities in their amino acid sequence. The most active of these substances is .beta.-endorphin, a polypeptide of 31 amino acids whose sequence (except for the c-terminal gistamine) is shown in FIG. 1.
In the mammalian pituitary, .beta.-endorphin is synthesized as a precursor protein of molecular weight about 30,000. The precursor protein includes the amino acid sequences of adrenocorticotropic hormone (ACTH), .beta.-lipotropin (.beta.-LPH), .alpha.-melanocyte stimulating hormone (.alpha.-MSH), .beta.-melanocyte stimulating hormone (.beta.-MSH), corticotrophin-like intermediate lobe peptide (CLIP), met-enkephalin and .beta.-endorphin. (See, e.g. Roberts, J. L. and Herbert, E., Proc. Natl. Acad. Sci. U.S. 74, 4826 (1977); Mains, E. and Eipper, B. A., J. Biol. Chem. 251, 4115 (1976); Nakanishi, S., et al., Proc. Natl. Acad. Sci. 73, 4319 (1976)). Normally, the precursor protein is processed by post-translational proteolysis and glycosylation, to generate the individual peptide hormones (Roberts, J. L., et al., Biochemistry 17, 3609 (1978); Eipper, B. A. and Mains, R. E., J. Biol. Chem. 253, 5732 (1978)).
To the extent the functions of the hormonal peptides contained within the precursor protein sequence are understood, they appear to be related generally to physical and biochemical adaptations to stress. The physiological responses to .beta.-endorphin appear to be analogous to the physiological effects of such opium alkaloids as morphine and heroin. Such effects are well known and include analgesia, altered emotional state, and reduced anxiety.
The medical uses of .beta.-endorphin are generally those for which the opiate alkaloids are presently employed. Therefore, the availability of an endogenous substance capable of producing the same effects is highly attractive to the medical profession. In addition, the expansion of medical uses for the opiates coupled with world population increase will lead to a projected world-wide shortage of morphine and its analogs within a few years. At the present time, .beta.-endorphin is known to be useful as an analgesic and in the treatment of intractable pain. Intra-cerebral administration of .beta.-endorphin, via an implanted delivery tube, has been employed in the successful treatment of such forms of intractable pain as phantom limb pain. In addition, .beta.-endorphin is effective as an intravenously administered analgesic. .beta.-endorphin has been reported to be effective in the treatment of certain mental disorders related to mood, affect and anxiety, including schizophrenia. It is also known that .beta.-endorphin binds to the opiate receptors in the gut so that it can be applied in the treatment of such gastrointestinal disorders as severe diarrhea, which presently are treated with opiates. It has also been shown that the administration of .beta.-endorphin induces increased plasma levels of prolactin and may therefore prove effective as a long-term birth control agent. Because of the presently known and predicted uses for .beta.-endorphin in medical care, there is presently a substantial market for the compound as an investigational drug. Prior to the present invention, the compound was obtainable in small quantity and great expense either by purification from brain extracts or by chemical synthesis. At current prices, the market for .beta.-endorphin as an investigational drug in the United State alone is estimated at several million dollars per year. Clearly, a method for producing .beta.-endorphin in quantity, with substantial economies of scale, is highly desirable. The present invention provides such a method.
Although .beta.-endorphin can be obtained from extracts of brain tissue, the yields are relatively small. Chemical synthesis has been achieved by Li, C.H., et al., Biochem. Biophys. Res. Commun. 71, 19 (1976). DNA sequences coding for all or part of the ACTH/.beta.-endorphin precursor protein have been cloned by the cDNA method (Roberts, J. L., et al., Proc. Natl. Acad. Sci. 76, 2153 (1979); Nakanishi, S., et al., Nature 278, 423 (1979)). The cloning of a DNA coding for a portion of the ACTH/.beta.-endorphin precursor protein is described in application Ser. No. 972,430, now U.S. Pat. No. 4,322,499, incorporated herein by reference. The genetic material cloned therein was employed as a starting material in the present invention.
Methods for the expression of heterologous DNA in a microorganism are now known. In principle, the heterologous DNA coding sequence is inserted in a DNA transfer vector at a point located within an expressible operon. The inserted sequence must be in reading frame phase with the coding sequence of the peron, and oriented in the same direction with respect to translation. When the conditions are met, translation of the operon results in "readthrough" to the inserted coding sequence such that the protein produced is a fusion protein comprising an N-terminal amino acid sequence coded by the expressible operon, followed by an amino acid sequence coded by the insert. See Polisky, B., et al, Proc. Natl. Acad. Sci., 73, 3900 (1976); Itakura, K., et al, Science, 198, 1056 (1977). Several expressible operons have been employed, including insertion in the .beta.-galactosidase gene, the .beta.-lactamase gene, and the tryptophan operon.
Although the genetic code is said to be "universal" in the sense that all known living organisms use the same code, it is well known that higher organisms, such as mammals, preferentially employ a set of codons which differs from that preferred by microorganisms such as bacteria. This observation has led investigators to choose synthetic coating sequences employing codons preferred by bacteria. Such a strategy does not appear to be necessary, at least for the expression of immunologically cross-reactive material, since both synthetic and naturally occurring sequences can be expressed in bacteria to yield an immunologically active product (Itakura, K., et al., Science 198, 1056 (1977); Material, J. A., et al., Science 205, 602 (1979)). Until the present invention, however, evidence of a biologically functional hormone has not been presented, either for synthetic or naturally occurring coding sequences. The present invention demonstrates the feasibility of using coding sequences comprising naturally occurring mammalian codons to achieve expression of biologically active protein by a recombinant microorganism.